Project 3 extends the investigations of nutritional modulation of gene expression and histopathology to human subjects. Experiments are carried out in the GCRC at Rockefeller University by a well-organized team of investigators. Subjects are recruited from a number of different registries, and used in crossover design studies to evaluate how individual nutritional factors can modulate profiles of gene expression in a rigorously controlled and monitored dietary environment. The first two protocols described compare: 1. high and low dietary calcium; 2. high dietary vitamin D on a high or low calcium background. Additional protocols will be developed based on the outcome of these studies, data from Project 1, and progress from a pilot project (Weber) that will develop a registry of genetically wellcharacterized subjects. Biopsies from subjects are evaluated by the Histopathology Core for alterations in cell proliferation, apoptosis, and lineage-specific differentiation. RNA is extracted for microarray analysis, using well established procedures, and a 27,000 member human cDNA array fabricated by the Albert Einstein Core facility. Analysis of the array data will be carried out by the Genomics and Biostatistics Cores, in collaboration, and will be instructed by the extensive data bases we will generate on nutritional - genetic interactions in mice in Project 1, and by microarray data bases we have already generated, both published and unpublished, that dissect pathways of cell intestinal cell maturation. Important changes in gene expression will be confirmed by a combination of Real-Time PCR and laser capture microdissection, and will be prioritized for pursuit of functional role by application of novel technologies in the Genomics Core, including imaging of transcription sites in situ and high-throughput structural proteomics. Core C will also use DNA from the biopsies and a high-throughput assay of genome-wide methylation to determine the extent to which alterations in gene expression are linked to altered locus methylation.